Naturally processed peptides derived from Toxoplasma gondii (T.gondii) -infected cells were acid extracted and detected by cytotoxic T lymphocytes (C) induced from peripheral blood lymphocytes of a patient with chronic toxoplasmosis. The CTL lines were obtained by weekly in vitro stimulation with a T.gondii-infected human B lymphoma line, ARH,which shares HLA-A2 and Cw4 determinants with the patient. The lytic activity of these CTL lines against T.gondii-infected ARH and ARH pulsed with fraction 29 of a reversed-phase high-performance liquid chromatography (RP-HPLC) extract from T.gondii-infected ARH was inhibited by an anti-HLA-A,B,C monoclonal antibody (mAb) and an anti-HLA-A2 mAb. Anti-HLA-DR mAb failed to block the lytic activity. Thus, the presentation of peptides by T.gondii-infected cells for CTL is mediated by HLA-A2 molecules. The peptides bound to HLA-A2 molecules were purified with an anti-HLA-A2 mAb-coupled immunoadsorbent column from T.gondii-infected ARH cell lysate. The
… Morepeptides dissociated from the complex of HLA-A2 molecules and fractionated by RP-HPLC in fraction 29 were submitted for sequential NH_2-terminal Edman degradation. The amino acid sequence analysis of fraction 29 revealedthat a dominant leucine was found at position 2, isoleucine was found at position 3, phenylalanine was found at position 8, and methionine and phenylalanine were dominant at position 9. Thus, the amino acid sequence of the HLA-A2-bound peptide in fraction 29 was in part consistent with the predictive algorithm of HLA-A2-binding peptide motifs.一方、トキソプラズマの細胞内抗原提示機構の分子論的解折を目的として、主要トキソプラズマ表面抗原と報告されているSAG1分子の真核細胞への遺伝子導入実験を開始した。SAG1分子はマウスに免疫することにより細胞傷害性T細胞が誘導され、トキソプラズマ感染に対する防御免疫が成立することが報告されている。トキソプラズマ遺伝子導入実験については、BoothroydがCHO細胞にSAG1遺伝子を導入し、B細胞エピトープ解析を進めているが、細胞内抗原処理・抗原提示能力をもつBリンパ腫などへの遺伝子導入実験には成功していない。我々はこれまでにヒトB細胞腫ARH(HLA-A2)、マウスTap-1ペプチドトランスポーター欠損変異細胞株RMA.S(H-2^b)へのSAG1遺伝子導入に成功し、遺伝子導入細胞がCTLの標的細胞となることを確認している。遺伝子導入はSAG1遺伝子プライマーを用いてPCRによりSAG1遺伝子を確認し、RT-PCRによりmRNAの発現を確認した。今後は、SAG1分子のヒト(HLA-A2)およびマウス(H-2^b)に結合するT細胞エピトープのアミノ酸配列を解析し、SAG1遺伝子導入細胞を用いて感染細胞では抗原量が微量なために解析が困難である細胞内抗原プロセッシング機構の解析を行う。 Less